Quinazoline inhibitors of activating mutant forms of epidermal growth factor receptor

ABSTRACT

The invention relates to compounds of formula (I), or a pharmaceutically acceptable salt thereof: 
                         
which possess inhibitory activity against activating mutant forms of EGFR, and are accordingly useful for their anti-cancer activity and in methods of treatment of the human or animal body. The invention also relates to processes for the manufacture of the compounds, or a pharmaceutically acceptable salt thereof, to pharmaceutical compositions containing them and to their use in the manufacture of medicaments of use in the production of an anti-cancer effect in a warm-blooded animal such as man.

This application is a continuation of U.S. patent application Ser. No.14/709,900, filed May 12, 2015, issuing, which is a continuation of U.S.patent application Ser. No. 14/197,476, filed Mar. 5, 2014 (now U.S.Pat. No. 9,066,979, issued Jun. 30, 2015), which claims priority under35 U.S.C. §119(a) (Pre-AlA) to International Application No.PCT/CN2013/072250, filed Mar. 6, 2013. The contents of the foregoingapplications are hereby incorporated in their entirety.

The present invention relates to certain4-(substituted-anilino)-6-O-(substituted-piperizine-carbonyl)quinazolinecompounds and pharmaceutically salts thereof which may be useful in thetreatment or prevention of a disease or medical condition mediatedthrough activating mutant forms of epidermal growth factor receptor(EGFR), for example the L858R activating mutant and/or the Exon 19deletion activating mutants. Such compounds and salts thereof may beuseful in the treatment or prevention of a number of different cancers.The invention also relates to pharmaceutical compositions comprisingsaid compounds, or a pharmaceutically salt thereof, crystalline forms ofthese compounds, or a pharmaceutically salt thereof, intermediatesuseful in the manufacture of said compounds, or a pharmaceutically saltthereof, and to methods of treatment of diseases mediated by activatingmutant forms of EGFR using said compounds, or a pharmaceutically saltthereof.

EGFR (otherwise known as ErbB1 or HER1) is a transmembrane proteintyrosine kinase member of the erbB receptor family. Upon binding of agrowth factor ligand such as epidermal growth factor (EGF), the receptorcan homo-dimerise with another EGFR molecule or hetero-dimerise withanother family member such as erbB2 (HER2), erbB3 (HER3), or erbB4(HER4).

Homo- and/or hetero-dimerisation of erbB receptors results in thephosphorylation of key tyrosine residues in the intracellular domain andleads to the stimulation of numerous intracellular signal transductionpathways involved in cell proliferation and survival. Deregulation oferbB family signalling promotes proliferation, invasion, metastasis,angiogenesis, and tumour cell survival and has been described in manyhuman cancers, including those of the lung, head and neck and breast.

The erbB family therefore represents a rational target for anticancerdrug development and a number of agents targeting EGFR or erbB2 are nowclinically available, including gefitinib (IRESSA™), erlotinib(TARCEVA™) and lapatinib (TYKERB™, TYVERB™). Detailed reviews of erbBreceptor signalling and its involvement in tumourigenesis are providedin New England Journal of Medicine [2008) Vol. 358; 1160-74 andBiochemical and Biophysical Research Communications [2004) Vol. 319:1-11.

In 2004 it was reported (Science [2004] Vol. 304: 1497-500 and NewEngland Journal of Medicine [2004] Vol. 350; 2129-39) that activatingmutations in EGFR correlated with response to gefitinib therapy innon-small-cell lung cancer (NSCLC). Approximately 90% of NSCLCassociated EGFR mutations consist of two major EGFR mutations(E746-A750del in Exon 19 and L858R substitution mutation in Exon 21)(Pao et al. Proceedings of the National Academy of Sciences of theUnited States of America [2004, Vol. 13: 306-11 and Kosada et al. CancerResearch [2004] Vol. 64: 8919-23). These activating mutations, result inan increase in affinity for small molecule tyrosine kinase inhibitorssuch as gefitinib and erlotinib and a decrease in affinity for adenosinetriphosphate (ATP) relative to wild type (WT) EGFR.

However, adverse effects, such as skin rash and diarrhoea, which areconsidered to be related to inhibition of WT EGFR signalling pathways innormal skin and gut cells, were reported in >60% NSCLC patients treatedwith gefitinib or erlotinib (Zhou C C et al. Journal of ClinicalOncology [2011], Vol. 12: 735-42; Mok T S et al. New England Journal ofMedicine [2009], Vol. 361: 947-57). In addition, both gefitinib anderlotinib showed limited effects on treating NSCLC patients with brainmetastasis, since neither of them effectively cross theblood-brain-barrier (BBB) (McKillop D et al. Xenobiotica [2004], Vol.34: 983-1000; Jackman D M et al. Journal of Clinical Oncology [2006],Vol. 24: 4517-20 Grommes C et al. Neuro-Oncology [2011], Vol. 13:1364-9), while several reports show that lung cancer brain metastasisare emerging as an unmet medical need (Gavrilovic et al, Journal ofNeurooncology [2005], Vol. 75: 5-14; Barnholtz-Sloan J S et al. Journalof Clinical Oncology. [2004], 22: 2865-72; Schouten L J et al, Cancer[2002], Vol. 94: 2698-705).

AstraZeneca has investigated sapitinib (AZD8931), an equipotentinhibitor of EGFR, HER2 and HER3 receptors, for use in breast cancer. Todate sapitinib has been studied in three phase II clinical trials; thefirst in combination with paclitaxel versus paclitaxel alone in advancedbreast cancer patients expressing low levels of HER2; the second incombination with anastrozole versus anastrozole alone in hormonereceptor positive advanced breast cancer; and the third in combinationwith paclitaxel versus paclitaxel alone in metastatic, gastric orgastro-oesophageal junction cancer who progress following first linetherapy and are ineligible for treatment with trastuzumab by HER2status. The compound of the present invention is structurally distinctfrom sapitinib, and possesses enhanced brain penetration propertieswhich makes it potentially useful in the treatment of cancers that havemetastasised to the brain.

Currently some irreversible EGFR inhibitors, such as afatinib anddacomitinib, are under clinical development. Although these compoundsshowed comparable effects on EGFR activating mutations in NSCLC patientswith gefitinib and erlotinib, they demonstrated more severe adverseeffects, such as skin rash (>90% skin rash and diarrhoea) (Zhou C C etal. Journal of Clinical Oncology [2011], Vol. 12: 735-42; Mok T S et al.New England Journal of Medicine [2009], Vol. 361: 947-57; Miller V A etal. Lancet Oncology [2012], Vol. 13: 528-38; Ramalingam S S et al.Journal of Clinical Oncology [2012], Vol. 30: 3337-44). The compounds ofthe present invention are reversible inhibitors, and are thereforeexpected to have less EGFR-related adverse effects than afatinib anddacomitinib.

Certain quinazoline compounds have been disclosed, e.g. “Preparation ofquinazoline derivatives for treatment of tumors” (US 20080177068 A1),“Preparation of quinazoline derivatives for treatment of tumors” (US20080167328 A1), “Preparation of saccharide derivatives of quinazolinesas protein tyrosine kinase inhibitors” (CN 101857618 A), “Preparation ofchlorofluoroanilinomethoxy-N-methylcarbamoylmethylpiperidinyloxyquinazolinederivatives for use as antitumor agents” (WO 2010061208 A2),“Preparation of 4-aminoquinazoline derivatives as antineoplastic agents(CN 101367793 A)”, “Preparation of proline quinazoline derivatives asantiproliferative agents (BR 2006002275 A)”, “Preparation of quinazolinederivatives as protein kinase inhibitors” (WO 2005097137 A2),“Preparation of quinazoline derivatives as protein kinase inhibitors”(WO 2005097134 A2), “Preparation of quinazoline derivatives as EGFRtyrosine kinase inhibitors” (WO 2005028469 A1), “Preparation ofphenylamino-substituted quinazolines as inhibitors of EGF and ErbB-2kinases” (WO 2005028470 A1), “Preparation of quinazoline derivatives asEGFR tyrosine kinase inhibitors” (WO 2005026156 A1), “Preparation ofpiperidyl-quinazoline derivatives as tyrosine kinase inhibitors for thetreatment of tumors” (WO 2005012290 A1), “Preparation of4-anilinoquinazolines as antiproliferative agents” (WO 2003082831 A1),“Preparation of aminoquinazolines as epidermal growth factor receptorsignal transduction inhibitors” (WO 2002018351 A1), “Preparation ofquinazolines as aurora 2 kinase inhibitors” (WO 2001021594 A1),“Quinazolines and other bicyclic heterocycles, pharmaceuticalcompositions containing these compounds as tyrosine kinase inhibitors,and processes for preparing them” (WO 2000055141 A1), “Preparation ofquinazoline derivatives and their receptor tyrosine kinase inhibitoryproperties” (WO 9738994 A1), “Quinazoline derivatives as antitumoragents” (WO 9730034 A1), “Preparation of haloanilinoquinazolines asClass I receptor tyrosine kinase inhibitors” (WO 9633980 A1) and“Quinazoline derivatives useful for treatment of neoplastic disease”(U.S. Pat. No. 5,457,105).

The compounds of the invention, or a pharmaceutically acceptable saltthereof, when compared with other clinically available EGFR inhibitors,exhibit certain improved properties e.g. higher BBB penetration (thusmaking them potentially useful for the treatment of cancers that havemetastasised to the brain); show better selectivity between WT EGFR andmutant EGFR (which may result in less treatment side effects of skinrash and diarrhoea); whilst maintaining equivalent or improved activityagainst activating mutant EGFR (e.g. EGFR L858R activating mutant and/orthe Exon 19 deletion activating mutants). Therefore, such compounds, ora pharmaceutically acceptable salt thereof, may be especially useful inthe treatment of disease states in which these activating mutations ofEGFR are implicated, for example in the treatment of cancer.

Accordingly, the present invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof.

The structures of the clinical compounds referred to above are asfollows:

A suitable pharmaceutically acceptable salt of a compound of theinvention is, for example, an acid-addition salt, for example aninorganic or organic acid, for example hydrochloric, hydrobromic,sulphuric, phosphoric, citric, L-tartaric, glycolic, fumaric, or maleicacid. A particular pharmaceutically acceptable salt of a compound of theinvention is a hydrochloric acid salt.

Salts of the compounds of formula (I) may be formed, for example, byreacting the compound of formula (I) with an amount of acid in a mediumsuch as one in which the salt precipitates or in an aqueous mediumfollowed by lyophilization.

The compounds of formula (I), or a pharmaceutically acceptable saltthereof, have a chiral centre. It is to be understood that the inventionencompasses all stereoisomers (enantiomers and diastereoisomers) of thecompounds of formula (I), or a pharmaceutically acceptable salt thereof,that possess activating mutant EGFR inhibitory activity. The inventionfurther relates to any and all tautomeric forms of the compounds offormula (I), or a pharmaceutically acceptable salt thereof, that possessactivating mutant EGFR inhibitory activity. In a further aspect of theinvention there is provided an enantiomer of formula (I), or apharmaceutically acceptable salt thereof, substantially free of anyother enantiomers. In a further aspect of the invention there isprovided the (R)-enantiomer of formula (I), or a pharmaceuticallyacceptable salt thereof, substantially free of any other enantiomers. Ina further aspect of the invention there is provided the (S)-enantiomerof formula (I), or a pharmaceutically acceptable salt thereof,substantially free of any other enantiomers.

In one embodiment of the invention where the mixture comprises unequalmolar proportions of enantiomers, the mixture may have an enantiomericexcess selected from >50%, >70%, >90% and >95%. Particularly the mixturemay have an enantiomeric excess >98%. More particularly the mixture mayhave an enantiomeric excess >99%. More particularly the mixture may havean enantiomeric excess >99.5%.

It is also to be understood that certain compounds of formula (I), or apharmaceutically acceptable salt thereof, can exist in solvated as wellas unsolvated forms such as, for example, hydrated forms. It is to beunderstood that the invention encompasses all such solvated forms whichpossess activating mutant EGFR inhibitory activity.

It is further to be understood that the invention encompasses allisotopic forms of the compounds described herein. For example hydrogenincludes deuterium and carbon includes ¹²C and ¹³C.

In another aspect of the invention, particular compounds of theinvention are any one of the Examples or a pharmaceutically acceptablesalt thereof.

In another aspect of the invention, particular compounds of theinvention are selected from:

-   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl    (2R)-2,4-dimethylpiperazine-1-carboxylate;-   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl    (2S)-2,4-dimethylpiperazine-1-carboxylate; and-   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (±)    2,4-dimethylpiperazine-1-carboxylate;    or a pharmaceutically acceptable salt thereof.

In another aspect of the invention, particular compounds of theinvention are selected from:

-   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl    (2R)-2,4-dimethylpiperazine-1-carboxylate;-   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl    (2S)-2,4-dimethylpiperazine-1-carboxylate; and-   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (±)    2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from a pharmaceutically acceptable salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate hydrochloride.

In another aspect of the invention, a particular compound of theinvention is selected from4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from a pharmaceutically acceptable salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2S)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2S)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from a pharmaceutically acceptable salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(−)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(−)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from a pharmaceutically acceptable salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(+)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(+)-2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from a pharmaceutically acceptable salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (±)2,4-dimethylpiperazine-1-carboxylate.

In another aspect of the invention, a particular compound of theinvention is selected from4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (±)2,4-dimethylpiperazine-1-carboxylate.

It is also to be understood that certain compounds of the invention, ora pharmaceutically acceptable salt thereof, may exist in certaincrystalline forms. In particular4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate, has been identified as havingseveral crystalline forms—particularly Form A, Form E, Form I and FormJ. In addition the hydrochloride salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate may also exist in crystallineform—particularly mono-HCl salt Form A₁. It is to be understood that thepresent invention encompasses all such crystalline forms of thecompounds of formula (I), or a pharmaceutically acceptable salt thereof,which possess activating mutant EGFR inhibitory activity.

4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form, Form A

Form A is characterised in providing at least one of the following 2θvalues measured using CuKa radiation: 23.3 and 14.3°. Form A ischaracterised in providing an X-ray powder diffraction pattern,substantially as shown in FIG. 1. Ten X-Ray powder diffraction peaks areshown in Table A:

TABLE A Ten X-Ray Powder Diffraction peaks for Form A Angle 2-Theta (2θ)Intensity % 23.3 100.00 14.3 83.70 9.4 78.08 18.6 61.70 16.3 60.41 21.539.61 12.4 38.89 26.1 38.18 19.8 35.71 27.4 31.12

According to the present invention there is provided a crystalline form,Form A, which has an X-ray powder diffraction pattern with at least twospecific peaks at about 2-theta=23.3° and 14.3°.

According to the present invention there is provided a crystalline form,Form A, which has an X-ray powder diffraction pattern with specificpeaks at about 2-theta=23.3, 14.3, 9.4, 18.6, 16.3, 21.5, 12.4, 26.1,19.8, 27.4°.

According to the present invention there is provided crystalline form,Form A which has an X-ray powder diffraction pattern substantially thesame as the X-ray powder diffraction pattern shown in FIG. 1.

According to the present invention there is provided a crystalline form,Form A, which has an X-ray powder diffraction pattern with at least twospecific peaks at 2-theta=23.3° and 14.3° wherein said values may beplus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form A, which has an X-ray powder diffraction pattern with specificpeaks at 2-theta=23.3, 14.3, 9.4, 18.6, 16.3, 21.5, 12.4, 26.1, 19.8,27.4° wherein said values may be plus or minus 0.2° 2-theta.

DSC analysis of Form A shows a melting endotherm with an onset of 192.4°C. and a peak at 195.8° C. (FIG. 2).

4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form, Form E

Form E is characterised in providing at least one of the following 2θvalues measured using CuKa radiation: 7.3 and 13.7°. Form E ischaracterised in providing an X-ray powder diffraction pattern,substantially as shown in FIG. 3. Nine X-Ray powder diffraction peaksare shown in Table B:

TABLE B Nine X-Ray Powder Diffraction peaks for Form E Angle 2-Theta(2θ) Intensity % 7.3 100.00 13.7 81.83 13.4 74.07 17.6 28.89 5.6 28.0210.8 19.08 21.7 19.04 26.5 17.10 28.4 13.41

According to the present invention there is provided a crystalline form,Form E, which has an X-ray powder diffraction pattern with at least twospecific peaks at about 2-theta=7.3° and 13.7°.

According to the present invention there is provided a crystalline form,Form E, which has an X-ray powder diffraction pattern with specificpeaks at about 2-theta=7.3, 13.7, 13.4, 17.6, 5.6, 10.8, 21.7, 26.5,28.4°.

According to the present invention there is provided crystalline form,Form E which has an X-ray powder diffraction pattern substantially thesame as the X-ray powder diffraction pattern shown in FIG. 3.

According to the present invention there is provided a crystalline form,Form E, which has an X-ray powder diffraction pattern with at least twospecific peaks at 2-theta=7.3° and 13.7° wherein said values may be plusor minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form E, which has an X-ray powder diffraction pattern with specificpeaks at 2-theta=7.3, 13.7, 13.4, 17.6, 5.6, 10.8, 21.7, 26.5, 28.4°wherein said values may be plus or minus 0.2° 2-theta.

DSC analysis of Form E shows a melting endotherm with an onset of 194.2°C. and a peak at 196.3° C. (FIG. 4).

4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form, Form I

Form I is characterised in providing at least one of the following 20values measured using CuKa radiation: 3.5 and 7.0°. Form I ischaracterised in providing an X-ray powder diffraction pattern,substantially as shown in FIG. 5. Ten X-Ray powder diffraction peaks areshown in Table C:

TABLE C Ten X-Ray Powder Diffraction peaks for Form I Angle 2-Theta (2θ)Intensity % 3.5 100.00 7.0 41.22 9.5 32.57 6.4 32.54 14.3 25.70 18.024.80 16.4 22.12 15.3 10.95 4.7 7.05 21.3 4.54

According to the present invention there is provided a crystalline form,Form I, which has an X-ray powder diffraction pattern with at leastthree specific peaks at about 2-theta 3.5°, 7.0° and 9.5°.

According to the present invention there is provided a crystalline form,Form I, which has an X-ray powder diffraction pattern with specificpeaks at about 2-theta=3.5, 7.0, 9.5, 6.4, 14.3, 18.0, 16.4, 15.3, 4.7,21.3°.

According to the present invention there is provided crystalline form,Form I which has a X-ray powder diffraction pattern substantially thesame as the X-ray powder diffraction pattern shown in FIG. 5.

According to the present invention there is provided a crystalline form,Form I, which has an X-ray powder diffraction pattern with at leastthree specific peaks at 2-theta=3.5°, 7.0° and 9.5° wherein said valuesmay be plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form I, which has an X-ray powder diffraction pattern with specificpeaks at 2-theta=3.5, 7.0, 9.5, 6.4, 14.3, 18.0, 16.4, 15.3, 4.7, 21.3°wherein said values may be plus or minus 0.2° 2-theta.

DSC analysis of Form I shows a melting endotherm with an onset of 193.3°C. and a peak at 195.9° C. (FIG. 6).

4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form, Form J

Form J is characterised in providing at least one of the following 20values measured using CuKa radiation: 7.8 and 7.0°. Form J ischaracterised in providing an X-ray powder diffraction pattern,substantially as shown in FIG. 7. Ten X-Ray powder diffraction peaks areshown in Table D:

TABLE D Ten X-Ray Powder Diffraction peaks for Form J Angle 2-Theta (2θ)Intensity % 7.8 100.00 7.0 49.36 4.9 45.57 15.9 27.11 17.7 20.89 3.417.30 20.7 16.71 9.8 14.59 13.9 14.11 12.7 10.83

According to the present invention there is provided a crystalline form,Form J, which has an X-ray powder diffraction pattern with at least twospecific peaks at about 2-theta=7.8° and 7.0°.

According to the present invention there is provided a crystalline form,Form J, which has an X-ray powder diffraction pattern with specificpeaks at about 2-theta=7.8, 7.0, 4.9, 15.9, 17.7, 3.4, 20.7, 9.8, 13.9,12.7°.

According to the present invention there is provided crystalline form,Form J which has an X-ray powder diffraction pattern substantially thesame as the X-ray powder diffraction pattern shown in FIG. 7.

According to the present invention there is provided a crystalline form,Form J, which has an X-ray powder diffraction pattern with at least twospecific peaks at 2-theta=7.8° and 7.0° wherein said values may be plusor minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form J, which has an X-ray powder diffraction pattern with specificpeaks at 2-theta=7.8, 7.0, 4.9, 15.9, 17.7, 3.4, 20.7, 9.8, 13.9, 12.7°wherein said values may be plus or minus 0.2° 2-theta.

DSC analysis of Form J shows a melting endotherm with an onset of 193.3°C. and a peak at 195.8° C. (FIG. 8).

4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate hydrochloride salt incrystalline form, mono-HCl salt Form A₁

Mono-HCl salt Form A₁ is characterised in providing at least one of thefollowing 2θ values measured using CuKa radiation: 12.3 and 13.9°.Mono-HCl salt Form A₁ is characterised in providing an X-ray powderdiffraction pattern, substantially as shown in FIG. 9. Nine X-Ray powderdiffraction peaks are shown in Table E:

TABLE E Nine X-Ray Powder Diffraction peaks for mono-HCl salt Form A₁Angle 2-Theta (2θ) Intensity % 12.3 100.00 13.9 40.45 9.3 29.34 23.326.42 18.7 20.54 16.0 17.94 24.6 10.24 26.8 8.94 28.0 7.90

According to the present invention there is provided a crystalline form,mono-HCl salt Form A₁ which has an X-ray powder diffraction pattern withat least two specific peaks at about 2-theta=12.3° and 13.9°.

According to the present invention there is provided a crystalline form,mono-HCl salt Form A₁ which has an X-ray powder diffraction pattern withspecific peaks at about 2-theta=12.3, 13.9, 9.3, 23.3, 18.7, 16.0, 24.6,26.8, 28.0°.

According to the present invention there is provided crystalline form,mono-HCl salt Form A₁ which has an X-ray powder diffraction patternsubstantially the same as the X-ray powder diffraction pattern shown inFIG. 9.

According to the present invention there is provided a crystalline form,mono-HCl salt Form A₁ which has an X-ray powder diffraction pattern withat least two specific peaks at 2-theta=12.3° and 13.9° wherein saidvalues may be plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,mono-HCl salt Form A₁ which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=12.3, 13.9, 9.3, 23.3, 18.7, 16.0, 24.6, 26.8,28.0° wherein said values may be plus or minus 0.2° 2-theta.

DSC analysis of mono-HCl salt Form A₁ shows a melting endotherm with anonset of 259.6° C. and a peak at 261.4° C. (FIG. 10).

LEGENDS TO FIGURES

FIG. 1: X-Ray Powder Diffraction Pattern of Form A

FIG. 2: DSC Thermogram of Form A

FIG. 3: X-Ray Powder Diffraction Pattern of Form E

FIG. 4: DSC Thermogram of Form E

FIG. 5: X-Ray Powder Diffraction Pattern of Form I

FIG. 6: DSC Thermogram of Form I

FIG. 7: X-Ray Powder Diffraction Pattern of Form J

FIG. 8: DSC Thermogram of Form J

FIG. 9: X-Ray Powder Diffraction Pattern of mono-HCl salt Form A₁

FIG. 10: DSC Thermogram of mono-HCl salt Form A₁

When it is stated that the present invention relates to a crystallineform, the degree of crystallinity is conveniently greater than about60%, more conveniently greater than about 80%, conveniently greater thanabout 90% and more conveniently greater than about 95%. Mostconveniently the degree of crystallinity is greater than about 98%.

It will be understood that the 2-theta values of the X-ray powderdiffraction pattern may vary slightly from one machine to another orfrom one sample to another, and so the values quoted are not to beconstrued as absolute. It is known that an X-ray powder diffractionpattern may be obtained which has one or more measurement errorsdepending on measurement conditions (such as equipment or machine used).In particular, it is generally known that intensities in an X-ray powderdiffraction pattern may fluctuate depending on measurement conditions.Therefore it should be understood that the polymorphic forms of thepresent invention are not limited to the crystals that provide X-raypowder diffraction patterns identical to the X-ray powder diffractionpattern shown in the figures, and any crystals providing X-ray powderdiffraction patterns substantially the same as those shown in thefigures fall within the scope of the present invention. A person skilledin the art of X-ray powder diffraction is able to judge the substantialidentity of X-ray powder diffraction patterns.

Persons skilled in the art of X-ray powder diffraction will realise thatthe relative intensity of peaks can be affected by, for example, grainsabove 30 microns in size and non-unitary aspect ratios, which may affectanalysis of samples. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values. (Jenkins, R & Snyder, R. L. ‘Introduction toX-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C. W. (1948),Chemical Crystallography, Clarendon Press, London; Klug, H. P. &Alexander, L. E. (1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is approximately plus or minus 0.2° 2-theta, and suchdegree of a measurement error should be taken into account whenconsidering the X-ray powder diffraction patterns shown in the figuresand tables. Furthermore, it should be understood that intensities mightfluctuate depending on experimental conditions and sample preparation(preferred orientation).

Therefore, in a further aspect of the invention there is provided4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form.

In a further aspect of the invention there is provided apharmaceutically acceptable salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form.

In a further aspect of the invention there is provided a hydrochloridesalt of 4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form A.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form E.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form I.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form J.

In one aspect of the invention, the hydrochloride salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of mono-HCl salt Form A₁.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form A and is substantially free of any other Forms.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form E and is substantially free of any other Forms.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form I and is substantially free of any other Forms.

In one aspect of the invention,4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of Form J and is substantially free of any other Forms.

In one aspect of the invention the hydrochloride salt of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate in crystalline form is in theform of mono-HCl salt Form A₁ and is substantially free of any otherForms.

The term “substantially free” refers to less than 10% of another Form orForms, enantiomer or enantiomers, particularly less than 5%. In anotheraspect “substantially free” refers to less than 1% of another Form orForms, enantiomer or enantiomers

As stated hereinbefore the compounds, or a pharmaceutically acceptablesalt thereof, defined in the present invention possess anti-canceractivity which is believed to arise from the activating mutant EGFRinhibitory activity, and other properties, of the compounds, or apharmaceutically acceptable salt thereof. These properties may beassessed, for example, using the procedures set out below.

Assay 1: Cellular Phosphorylation Assay

The human lung cell line NCI-H3255 (L858R) was obtained from theAmerican Type Culture Collection. The NCI-H3255 cells were maintained inBEBM media (Lonza; CC-3171), containing 10% fetal bovine serum (FBS)(Gibco; 10099-141), supplemented with BEGM kit (Lonza; CC-4175). Thehuman lung cell line PC-9 (Exon 19 deletion EGFR) was obtained from theAmerican Type Culture Collection. PC-9 cells were maintained in RPMI1640 (Gibco; 22400-089), containing 10% fetal bovine serum. The humanlung cell line NCI-H838 (EGFR wild type) was obtained from the AmericanType Culture Collection. NCI-H838 cells were maintained in RPMI 1640(Gibco; 22400-089), containing 10% fetal bovine serum.

All cells were grown in a humidified incubator at 37° C. with 5% CO₂.Assays to measure cellular phosphorylation of endogenous p-EGFR in celllysates were carried out according to the protocol described in thePathScan® Phospho-EGF Receptor (Tyr1068) Sandwich ELISA Kit (CellSignalling kit catalogue number #7240).

100 μL of cells were seeded (32000 cells/well) in RPMI 1640+1% fetalbovine serum in Corning Costar, 96 well cell culture plates andincubated at 37° C. with 5% CO₂ overnight. Cells were acoustically dosedusing a Tecan, with compounds serially diluted in 100% DMSO. Cell plateswere incubated for a further 4 h after the compounds were added, (forNCI-H838: rhEGF (R&D catalogue number#236-EG) was added to cell platewith final concentration 100 ng/ml rhEGF to stimulate 5 minutes), thenfollowing aspiration of medium, 110 μL IP lysis buffer (IP lysis buffer:add 1:100 phosphatase inhibitor cocktail 2&3 (Sigma catalogue numberP5726&P0044), 1:100 protease inhibitor cocktail (Sigma catalogue numberP8340) to Pierce IP lysis buffer (Thermo catalogue number #87788)) wasadded to each well. The plates were put at 4° C. with rotation 300 rpmfor 0.5-1 hour. 100 μl/well of cell lysis was transferred to coatedplates (Cell Signalling kit catalogue number#7240) and incubatedovernight at 4° C. with rotation 300 rpm. The plates we taken from 4° C.to 37° C. with rotation 300 rpm for 1 hour. Following aspiration andwashing of the plates with 1× wash buffer, 100 μl of detection antibody(Cell Signalling kit catalogue number#7240) was added to each well. Theplate was sealed with tape and incubated for 2 hours at 37° C. withrotation 300 rpm. Following aspiration and washing of the plates with 1×wash buffer, 100 μl of HRP-linked secondary antibody (Cell Signallingkit catalogue number#7240) was added to each well. The plate was sealedwith tape and incubated for 1 hour at 37° C. with rotation 300 rpm.Following aspiration and washing of the plates with 1× wash buffer, 100μl of TMB substrate (Cell Signalling kit, catalogue number#7240) wasadded to each well. The plate was sealed with tape and incubated for 30minutes at 37° C. with 300 rpm. 100 μl stop solution (Cell Signallingkit catalogue number#7240) was added to the plates and absorbance readat 450 nm within 30 minutes on SpectraMax M5e plate reader.

The data obtained with each compound was exported into a suitablesoftware package (such as H-BASE) to perform curve fitting analysis.From this data an 1050 value was determined by calculation of theconcentration of compound that is required to give a 50% effect.

The assay data (μM) in Assay 1 for the Examples of this application aswell as that obtained for gefitinib and erlotinib are shown in the tablebelow:

IC₅₀ IC₅₀ IC₅₀ Compound (NCI-H3255) (PC-9) (NCI-H838) Example 1 0.0050.006 0.06 Example 3 0.005 0.008 0.04 Example 4 0.001 0.004 0.04gefitinib 0.006 0.007 0.03 erlotinib 0.009 0.006 0.03

This shows that Example 1, Example 2, and Example 3 have comparablepotency to gefitinib and erlotinib.

Assay 2: Brain Blood Barrier Penetration Assay

It is believed that Kp,uu, the relationship between concentrations ofunbound drug in brain and in blood, is the key to prediction of CNSaction and should be the main parameter measured and optimized for indrug discovery (Di L et al., Journal of Medicinal Chemistry [2013], 56:2-12).

In vitro blood and brain binding assay was carried out on a HT-Dialysisplate (Gales Ferry, Conn.) with semi-permeable membrane. Diluted blood(1:1 with DPBS pH7.4) and brain homogenate (1:3 with DPBS pH7.4) werespiked with 5 μM test compound (in triplicate) and dialyzed againstequal volume of 150 μL 100 mM PBS buffer (pH7.4) at 37° C. for 4 hoursin a slowly rotated plate. At the end of incubation, a 50 μL aliquotfrom the receiver side and a 5 μL from the donor chamber were taken. The5 μL sample was further diluted with 45 μL of blank blood or brainhomogenate. Paired samples were matrix-matched with either buffer orblank blood/brain homogenate and mixed for 2 min, and then precipitatedwith 150 μL cold acetonitrile with 100 ng/mL tolbutamide as internalstandard. After centrifuging at 4000 rpm for 20 min, supernatant wasdiluted with 0.1% formic acid aqueous solution and analyzed for LC/MS/MS(API 4000, Applied Biosystems, Foster City). Unbound fraction (fu) oftest compound in the brain homogenate and diluted blood were calculatedby the ratio of the buffer side response to the brain homogenate/bloodside response, and unbound fraction (f_(u,bl) and f_(u,br)) of testcompound in non-diluted blood and tissue were calculated from measuredfu in homogenate and diluted blood with the following equation: f_(u,bl)(f_(u,br))=(1/D)/[(1/fu−1)+1/D)]. D is dilution factor.

A Short oral absorption (SOA) model is an in-vivo screening model toidentify brain penetration of a compound. Six male Han Wistar ratspurchased from Beijing Vital River were orally dosed with the compoundat 2 mg/kg in 1% methylcellulose. At 0.25, 0.5, 1, 2, 4 and 7 hourpost-dose, cerebral spinal fluid (CSF) was collected from cisternamagna, and blood samples (>60 μL/time point/each site) were collectedvia cardiac puncture, into separate EDTA coagulated tubes, and thenimmediately diluted with 3-fold volume of water. Brain tissue washarvested and homogenized in 3× volume of 100 mM phosphate bufferedsaline (pH7.4). All samples were stored at ˜−70° C. prior to LC/MS/MSanalysis.

Standards were prepared by spiking blank blood, brain homogenate andartificial CSF covering 0.2 to 500 ng/mL. Homogenized brain tissue alongwith blood samples were precipitated by adding 3-fold volume of coldacetonitrile containing internal standard (40 ng/mL Dexamethasone and 40ng/mL Diclofenac), and 10 μL of CSF samples were precipitated with 100μL of cold acetonitrile containing internal standard. After 2 min vortexand 5 min centrifugation at 14,000 rpm, supernatant was analyzed byLC/MS/MS (API 4000, Applied Biosystems, Foster City). Two sets ofstandard curves were run at the beginning and end of each batch fromblood sample analysis. For brain and CSF samples, one standard curve wasanalyzed along with test samples.

Total brain levels, expressed as brain/blood ratio (Kp) were measured byAUCbrain/AUCblood in rodents after oral administration. Free fraction oftest compound in biological matrix was determined by in vitro blood andbrain binding assay. Kp,uu was calculated by the following equation:Kp,uu=AUC (brain)/AUC (blood)×(f_(u,brain)/f_(u.blood)).

The assay data in Assay 2 for the Examples of this application as wellas data obtained for sapitinib (freebase form) is shown in the tablebelow:

Compound Kp, uu Example 1 1.3 Example 3 1.6 sapitinib 0.12demonstrating the superior brain barrier penetration properties of thecompounds of the present invention, when compared to sapitinib.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of formula (I), ora pharmaceutically acceptable salt thereof, as defined hereinbefore, inassociation with a pharmaceutically-acceptable diluent or carrier.

The composition may be in a form suitable for oral administration, forexample as a tablet or capsule, for parenteral injection (includingintravenous, subcutaneous, intramuscular, intravascular or infusion) asa sterile solution, suspension or emulsion, for topical administrationas an ointment or cream or for rectal administration as a suppository.Particularly the composition may be in a form suitable for oraladministration.

In general the above compositions may be prepared in a conventionalmanner using conventional excipients.

The compound of formula (I), or a pharmaceutically acceptable saltthereof, will be administered to a warm-blooded animal at a unit dosewithin the range 0.01-2000 mg/kg, particularly 2.5-1000 mg/kg,particularly 5-500 mg/kg, and this should provide a therapeuticallyeffective dose. However the daily dose will necessarily be varieddepending upon the host treated, the particular route of administration,and the severity of the illness being treated. Accordingly the optimumdosage may be determined by the practitioner who is treating anyparticular patient.

According to a further aspect of the present invention there is provideda compound of formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use in a method of treatment of thehuman or animal body by therapy.

As a result of its activating mutant EGFR inhibitory activity, thecompounds of formula (I), or a pharmaceutically acceptable salt thereof,are expected to be useful in the treatment of diseases or medicalconditions mediated alone or in part by activating mutant EGFR, forexample cancer. The types of cancers which may be susceptible totreatment using the compounds of formula (I), or a pharmaceuticallyacceptable salt thereof, include, but are not limited to, ovariancancer, cervical cancer, colorectal cancer, breast cancer, pancreaticcancer, glioma, glioblastoma, melanoma, prostate cancer, leukaemia,lymphoma, non-Hodgkins lymphoma, lung cancer, hepatocellular cancer,gastric cancer, gastrointestinal stromal tumour, thyroid cancer, bileduct cancer, endometrial cancer, renal cancer, anaplastic large celllymphoma, acute myeloid leukaemia, multiple myeloma, melanoma andmesothelioma. In a particular embodiment of the invention, the type ofcancer which may be susceptible to treatment using the compound offormula (I), or a pharmaceutically acceptable salt thereof isnon-small-cell lung cancer (NSCLC). In a further particular embodimentthe NCSLC cells in the warm blooded animal possess or have previouslybeen shown to possess activation mutations in the EGFR gene.

The compound of formula (I), or a pharmaceutically acceptable saltthereof, is useful in the treatment of disease states in whichactivating mutant EGFR is implicated. In one aspect of the inventionwhere activating mutant EGFR is referred to this refers one or moremutations in the ATP-binding site (kinase domain) of the EGFR gene,particularly around Exons 18-21, such as those described in WO2005/094357. In one aspect of the invention where activating mutant EGFRis referred to this refers to L858R activating mutant EGFR and/or Exon19 deletion activating mutant EGFR. In one aspect of the invention whereactivating mutant EGFR is referred to this refers to L858R activatingmutant EGFR and Exon 19 deletion activating mutant EGFR. In one aspectof the invention where activating mutant EGFR is referred to this refersto L858R activating mutant EGFR. In another aspect of the inventionwhere activating mutant EGFR is referred to this refers to Exon 19deletion activating mutant EGFR.

It is envisaged that for the methods of treatment of cancer mentionedherein, the compounds of formula (I), or a pharmaceutically acceptablesalt thereof, will be administered to a mammal, more particularly ahuman being. Similarly, the uses of the compounds of formula (I), or apharmaceutically acceptable salt thereof, for the treatment of cancermentioned herein, it is envisaged that the compounds of formula (I), ora pharmaceutically acceptable salt thereof, will be administered to amammal, more particularly a human being.

According to another aspect of the invention, there is thereforeprovided the compounds of formula (I), or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use as a medicament.

According to a further aspect of the invention there is provided the useof a compound of formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament forthe inhibition of activating mutant EGFR in a warm-blooded animal suchas man.

According to this aspect of the invention there is provided the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,as defined hereinbefore in the manufacture of a medicament for theproduction of an anti-cancer effect in a warm-blooded animal such asman.

According to a further feature of the invention, there is provided theuse of a compound of formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the treatment of ovarian cancer, cervical cancer, colorectalcancer, breast cancer, pancreatic cancer, glioma, glioblastoma,melanoma, prostate cancer, leukaemia, lymphoma, non-Hodgkins lymphoma,lung cancer, hepatocellular cancer, gastric cancer, gastrointestinalstromal tumour, thyroid cancer, bile duct cancer, endometrial cancer,renal cancer, anaplastic large cell lymphoma, acute myeloid leukaemia,multiple myeloma, melanoma and mesothelioma.

According to a further feature of the invention, there is provided theuse of a compound of formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the treatment of NSCLC.

According to a further feature of this aspect of the invention there isprovided a method of inhibiting activating mutant EGFR in a warm-bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of this aspect of the invention there isprovided a method for producing an anti-cancer effect in a warm-bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of this aspect of the invention there isprovided a method for producing an anti-cancer effect in a warm-bloodedanimal, such as man, in need of such treatment which comprises (1)determining whether or not the warm blooded animal has an activatingEGFR mutation in the tumour cell and (2) and if so administering to saidanimal an effective amount of the compound of formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore.

According to an additional feature of this aspect of the invention thereis provided a method of treating ovarian cancer, cervical cancer,colorectal cancer, breast cancer, pancreatic cancer, glioma,glioblastoma, melanoma, prostate cancer, leukaemia, lymphoma,non-Hodgkins lymphoma, lung cancer, hepatocellular cancer, gastriccancer, gastrointestinal stromal tumour, thyroid cancer, bile ductcancer, endometrial cancer, renal cancer, anaplastic large celllymphoma, acute myeloid leukaemia, multiple myeloma, melanoma andmesothelioma, in a warm-blooded animal, such as man, in need of suchtreatment which comprises administering to said animal an effectiveamount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, as defined hereinbefore.

According to an additional feature of this aspect of the invention thereis provided a method of treating NSCLC, in a warm-blooded animal, suchas man, in need of such treatment which comprises administering to saidanimal an effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention there is provided acompound of formula (I), or a pharmaceutically acceptable salt thereof,as defined hereinbefore for use in inhibiting activating mutant EGFR ina warm-blooded animal such as man.

According to this aspect of the invention there is provided a compoundof formula (I), or a pharmaceutically acceptable salt thereof, asdefined hereinbefore for use in the production of an anti-cancer effectin a warm-blooded animal such as man.

According to a further feature of the invention, there is provided acompound of formula (I), or a pharmaceutically acceptable salt thereof,as defined hereinbefore for use in the treatment of ovarian cancer,cervical cancer, colorectal cancer, breast cancer, pancreatic cancer,glioma, glioblastoma, melanoma, prostate cancer, leukaemia, lymphoma,non-Hodgkins lymphoma, lung cancer, hepatocellular cancer, gastriccancer, gastrointestinal stromal tumour, thyroid cancer, bile ductcancer, endometrial cancer, renal cancer, anaplastic large celllymphoma, acute myeloid leukaemia, multiple myeloma, melanoma andmesothelioma.

According to a further feature of the invention, there is provided acompound of formula (I), or a pharmaceutically acceptable salt thereof,as defined hereinbefore for use in the treatment of NSCLC.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore inassociation with a pharmaceutically-acceptable diluent or carrier foruse in inhibiting activating mutant EGFR in a warm-blooded animal suchas man.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore inassociation with a pharmaceutically-acceptable diluent or carrier foruse in the production of an anti-cancer effect in a warm-blooded animalsuch as man.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore inassociation with a pharmaceutically-acceptable diluent or carrier foruse in the treatment of ovarian cancer, cervical cancer, colorectalcancer, breast cancer, pancreatic cancer, glioma, glioblastoma,melanoma, prostate cancer, leukaemia, lymphoma, non-Hodgkins lymphoma,lung cancer, hepatocellular cancer, gastric cancer, gastrointestinalstromal tumour, thyroid cancer, bile duct cancer, endometrial cancer,renal cancer, anaplastic large cell lymphoma, acute myeloid leukaemia,multiple myeloma, melanoma and mesothelioma in a warm-blooded animalsuch as man.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore inassociation with a pharmaceutically-acceptable diluent or carrier foruse in the treatment of NSCLC in a warm-blooded animal such as man.

In any of the aspects or embodiments mentioned herein where cancer ismentioned said cancer may be selected from ovarian cancer, cervicalcancer, colorectal cancer, breast cancer, pancreatic cancer, glioma,glioblastoma, melanoma, prostate cancer, leukaemia, lymphoma,non-Hodgkins lymphoma, lung cancer, hepatocellular cancer, gastriccancer, gastrointestinal stromal tumour, thyroid cancer, bile ductcancer, endometrial cancer, renal cancer, anaplastic large celllymphoma, acute myeloid leukaemia, multiple myeloma, melanoma andmesothelioma.

In any of the aspects or embodiments mentioned herein where cancer ismentioned, particularly said cancer may be selected from lung cancer. Ina further aspect, particularly said cancer may be selected fromnon-small-cell lung cancer. In a further aspect, particularly saidcancer may be selected from non-metastatic non-small-cell lung cancer.In a further aspect, particularly said cancer may be selected frommetastatic non-small-cell lung cancer.

The compound of the present invention may be applied in the adjuvantand/or 1^(st) line and/or 2^(nd) line treatment settings of NSCLCpatients carrying activating mutant EGFR, with and without brainmetastasis.

In another aspect the cancer is in a non metastatic state.

In another aspect the cancer is in a metastatic state.

Certain NSCLC patients with brain metastasis exhibit CNS symptoms, suchas headache and vomiting. For these patients, whole brain radiationtherapy (WBRT) may be used to improve these symptoms. The compound ofthe present invention may be able to enhance the anti-tumour effect ofWBRT as well as to further improve CNS symptoms when used in combinationwith WBRT.

Herein, where the term “combination” is used it is to be understood thatthis refers to simultaneous, separate or sequential administration. Inone aspect of the invention “combination” refers to simultaneousadministration. In another aspect of the invention “combination” refersto separate administration. In a further aspect of the invention“combination” refers to sequential administration. Where theadministration is sequential or separate, the delay in administering thesecond component should not be such as to lose the beneficial effect ofthe combination.

In addition to their use in therapeutic medicine, the compounds offormula (I), or a pharmaceutically acceptable salt thereof, are alsouseful as pharmacological tools in the development and standardisationof in vitro and in vivo test systems for the evaluation of the effectsof activating mutant EGFR inhibitory activity in laboratory animals suchas cats, dogs, rabbits, monkeys, rats and mice, as part of the searchfor new therapeutic agents.

In the above other pharmaceutical composition, process, method, use andmedicament manufacture features, the alternative and preferredembodiments of the compounds of the invention described herein alsoapply.

EXAMPLES

The invention will now be illustrated in the following Examples inwhich, generally:

(i) in general, the course of reactions were followed by liquidchromatography mass spectrometry (LCMS) or thin later chromatography(TLC); the reaction times that are given are not necessarily the minimumattainable;

(ii) when necessary, organic solutions were dried over anhydrousmagnesium sulfate or anhydrous sodium sulfate, work-up procedures werecarried out using traditional layer separating techniques, evaporationswere carried out either by rotary evaporation under reduced pressure orin a Genevac HT-4/EZ-2.

(iii) yields, where present, are not necessarily the maximum attainable,and when necessary, reactions were repeated if a larger amount of thereaction product was required;

(iv) in general, the structures of the end-products were confirmed bynuclear magnetic resonance (NMR) and/or mass spectral techniques;electrospray mass spectral data were obtained using a Waters ZMD orWaters ZQ LC/mass spectrometer acquiring both positive and negative iondata, generally, only ions relating to the parent structure arereported; proton NMR chemical shift values were measured on the deltascale at 400 MHz using a Bruker NMR spectrometer or a Varian NMRspectrometer. The following abbreviations have been used: s, singlet; d,doublet; pd, partial doublet; t, triplet; q, quartet; m, multiplet; br,broad. Exchangeable protons are not always observed or reported in theNMR of end-products due to exchange with deuterated solvent oradvantageous deuterated water in the solvent or the signal is poorlyresolved and/or very broad;

(v) intermediates were not necessarily fully purified but theirstructures and purity were assessed by TLC, analytical HPLC and/or NMRanalysis;

(vi) unless otherwise stated, column chromatography (by the flashprocedure) and medium pressure liquid chromatography (MPLC) wereperformed on Merck Kieselgel silica (Art. 9385) or by using pre-packedsilica cartridges on semi-automated flash chromatography equipment (forexample a CombiFlash Companion); and

(vii) the following abbreviations have been used:

-   -   is Boc tert-butyloxycarbonyl;    -   CD₃OD deuteromethanol;    -   DMSO-d₆ hexadeuterodimethylsulfoxide;    -   CDCl₃ deuterochlorform;    -   PE petroleum ether;    -   IPA isopropanol;    -   iPrOAc isopropyl acetate;    -   MTBE methyl tert-butyl ether;    -   DCM dichloromethane;    -   THF tetrahydrofuran;    -   RT room temperature;    -   MeOH methanol;    -   EtOH ethanol; and    -   EtOAc ethyl acetate.        X-Ray Powder Diffraction

Analytical Instrument: Panalytical Empyrean. The X-ray powderdiffractogram was determined by mounting a sample of the crystallinematerial on a Si single crystal holder and spreading out the sample intoa thin layer with the aid of a microscope slide. The 2θ position wascalibrated against Panalytical 640 Si powder standard. The sampleirradiated with X-rays generated by a copper long-fine focus tubeoperated at 45 kV and 40 mA with a wavelength of Kα1=1.540598 angstromsand Kα2=1.544426 angstroms (Kα2/Kα1 intensity ratio is 0.50). Thecollimated X-ray source was passed through an programmed divergence slitset at 10 mm and the reflected radiation directed through a 5.5 mmantiscatter slit. The sample was exposed for 12.7 seconds per 0.0167°2-theta increment (continuous scan mode) over the range 3 degrees to 40degrees 2-theta in theta-theta mode. The running time was 3 minutes and57 seconds. The instrument was equipped with a RTMS detector(X'Celerator). Control and data capture was by means of a Dell Optiplex780 XP operating with data collector software. Persons skilled in theart of X-ray powder diffraction will realize that the relative intensityof peaks can be affected by, for example, grains above 30 microns insize and non-unitary aspect ratios that may affect analysis of samples.The skilled person will also realize that the position of reflectionscan be affected by the precise height at which the sample sits in thediffractometer and the zero calibration of the diffractometer. Thesurface planarity of the sample may also have a small effect. Hence thediffraction pattern data presented are not to be taken as absolutevalues.

Differential Scanning Calorimetry

Analytical Instrument: TA Instruments Q200 or Q2000 DSC. Typically lessthan 5 mg of material contained in a standard aluminium pan fitted witha lid was heated over the temperature range 25° C. to 300° C. at aconstant heating rate of 10° C. per minute. A purge gas using nitrogenwas used—flow rate 50 ml per minute.

Intermediate 1 5-Hydroxy-4-methoxy-2-nitrobenzoic acid

4,5-Dimethoxy-2-nitrobenzoic acid (145 g, 0.639 mol) was dissolved in asolution of sodium hydroxide (6N, 600 mL) and heated at 100° C. for 3 h.The mixture was cooled to RT, and poured into a mixture of concentratedhydrochloric acid and crushed ice (pH<2). The mixture was filtered, andthe filter cake was dried to give Intermediate 1 (149 g, crude) as ayellow solid, which was used without further purification. ¹H NMR(DMSO-d₆ 400 MHz): δ7.34 (s, 1H), 6.89 (s, 1H), 3.80 (s, 3H).

Intermediate 2 2-Amino-5-hydroxy-4-methoxybenzoic acid

A mixture of Intermediate 1 (50 g, 93.85 mmol) and 10% Pd/C (5 g) inMeOH (1.2 L) was stirred under H₂ atmosphere (50 psi) at RT for 4 h. Themixture was filtered and washed with MeOH (10×1 L). The combined MeOHextracts were concentrated to afford Intermediate 2 (27.7 g, 64% yield)as black solid which was used without further purification.

Intermediate 3 7-Methoxyquinazoline-4,6-diol

To a suspension of Intermediate 2 (88 g, 0.48 mol) in 2-methoxyethanol(2 L) was added formamidine (101 g, 0.96 mol) and the reaction mixturewas refluxed overnight. The reaction mixture was concentrated, dilutedwith water (1.5 L) and neutralized (to pH=7) with ammonia. The mixturewas filtered and the precipitate was washed with water. The precipitatewas dried under reduced pressure to afford Intermediate 3 as a brownsolid (62 g, 67% yield). ¹H NMR (DMSO-d₆ 400 MHz): δ 7.89 (s, 1H), 7.36(s, 1H), 7.08 (s, 1H), 3.88 (s, 3H).

Intermediate 4 4-Hydroxy-7-methoxyquinazolin-6-yl acetate

To a suspension of Intermediate 3 (52 g, 0.27 mol) and pyridine (53.6 g,0.68 mol) in anhydrous DCM (1 L) was added acetic chloride (52.9 g, 0.68mol) drop-wise and the mixture was stirred overnight at RT. The mixturewas poured into water (1 L) and extracted with DCM several times. Thecombined organic layers were washed with brine, dried over Na₂SO₄,concentrated to afford Intermediate 4 as a black solid (63.2 g, 100%yield). ¹H NMR (DMSO-d₆ 400 MHz): δ 8.62 (s, 1H), 7.88 (s, 1H), 7.37 (s,1H), 3.95 (s, 3H), 2.74 (s, 3H).

Intermediate 5 4-Chloro-7-methoxyquinazolin-6-yl acetate

A suspension of Intermediate 4 (75.6 g, 0.323 mol) in POCl₃ (287 mL) washeated to refluxed for 0.5 h. The reaction mixture was concentrated anddiluted with DCM (500 mL), poured into water (500 mL), filtered andwashed with DCM. The combined organic layers were washed with brine,dried over Na₂SO₄ and concentrated. Purification by chromatography(PE/EtOAc=1/1) gave Intermediate 5 (55 g, 67% yield) as white solid.

¹H NMR (CDCl₃ 400 MHz): δ 8.95 (s, 1H), 7.90 (s, 1H), 7.43 (s, 1H), 4.02(s, 1H), 2.39 (s, 1H).

Intermediate 64-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl acetate

To a suspension of Intermediate 5 (100 g, 0.396 mol) in acetonitrile (4L) was added 2-fluoro-3-chloroaniline (60.5 g, 0.416 mol) and thereaction mixture was heated to 80° C. overnight. The precipitate wascollected by filtration and dried in vacuo to afford Intermediate 6 (181g, 80% purity) as white solid which was used for next step directlywithout purification. ¹H NMR (DMSO-d₆ 400 MHz): δ 8.93 (s, 1H), 8.82 (s,1H), 7.67-7.63 (m, 1H), 7.59 (s, 1H), 7.56-7.52 (m, 1H), 7.39-7.35 (m,1H), 4.02 (s, 3H), 2.39 (s, 3H).

Intermediate 74-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-ol

To a solution of Intermediate 6 (181 g, 0.396 mol) in MeOH (2 L) wasadded potassium carbonate (138 g, 1 mol) and the reaction mixture wasstirred at RT overnight. The reaction mixture was filtered and the solidwashed with MeOH. The filtrate was concentrated in vacuo to affordIntermediate 7 (280 g, 60% purity, contained potassium carbonate). ¹HNMR (DMSO-d₆ 400 MHz): δ 8.01 (s, 1H), 7.61-7.58 (m, 1H), 7.27-7.24 (m,1H), 7.17-7.13 (m, 1H), 6.95 (s, 1H), 6.83 (s, 1H), 3.79 (s, 3H).

Intermediate 8 tert-Butyl(3R)-4-(chlorocarbonyl)-3-methylpiperazine-1-carboxylate

To a mixture of triphosgene (23 g, 75 mmol) in anhydrous DCM (250 mL)was added pyridine (18 g, 225 mmol) drop-wise followed by addition oftert-butyl (3R)-3-methylpiperazine-1-carboxylate (15 g, 75 mmol) at 0°C. The mixture was stirred overnight at RT. TLC showed the startingmaterial had been consumed. The mixture was concentrated to affordIntermediate 8 as yellow solid, which was used without furtherpurification.

Intermediate 9 4-tert-Butyl1-{4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl}(2R)-2-methylpiperazine-1,4-dicarboxylate

A mixture of Intermediate 7 (19.2 g, 60 mmol), Intermediate 8 preparedaccording to the above procedure and potassium carbonate (16.6 g, 120mmol) in anhydrous DMF (300 mL) was stirred overnight at RT. Thereaction mixture was poured into water (250 mL) and filtered, and thefilter cake was dried under vacuum to afford Intermediate 9 (25 g, 75%yield) as yellow solid. HPLC: t_(R)=2.68 min in 10-80AB_6 minchromatography (Ultimate XB-C18, 3.0*50 mm, 3 um). LCMS: t_(R)=0.792 minin 5-95AB_1.5 min chromatography (Welch Xtimate C18, 2.1*30 mm, 3 um),MS (ESI) m/z 546.0 [M+H]⁺.

Intermediate 104-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2-methylpiperazine-1-carboxylate

A mixture of Intermediate 9 (8.3 g, 15 mmol) in DCM (100 mL) andHCl/dioxane (10 mL, 4M) was stirred for 30 min at RT. After filtration,the solid was collected and redissolved in water, and then adjusted topH=8 with saturated NaHCO₃. The precipitate was collected and washedwith CH₂Cl₂. The solid was dried under vacuum to give the Intermediate10 (8 g, 85% purity) as yellow solid. This crude product was used forthe next step without purification.

Intermediate 11 (S)-2,4-dimethylpiperazine-1-carbonyl chloride

To a solution of triphosgene (1.04 g, 3.5 mmol) in DCM (20 mL) undernitrogen was added pyridine (2.3 g, 28.0 mmol) drop-wise at 0° C.followed by addition of (S)-1,3-dimethylpiperazine (800 mg, 7.0 mmol) inDCM (30 mL), the reaction mixture was warmed to RT and stirred overnightas monitored by TLC (R_(f)=0.9, PE: EtOAc=1:1). The mixture wasconcentrated to give Intermediate 11 (3 g, crude) which was used withoutpurification.

Intermediate 12 (±)-tert-Butyl(4-(chlorocarbonyl)-3-methylpiperazine-1-carboxylate

To a mixture of triphosgene (23 g, 75 mmol) in anhydrous DCM (250 mL)was added pyridine (18 g, 225 mmol) drop-wise followed by addition of(±)-tert-butyl 3-methylpiperazine-1-carboxylate (15 g, 75 mmol) at 0° C.The mixture was stirred overnight at RT. TLC showed the startingmaterial was consumed. The mixture was concentrated to affordIntermediate 12 as yellow solid, which was used without furtherpurification.

Intermediate 13 (±)-4-tert-Butyl1-{4-[(2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl}2-methylpiperazine-1,4-dicarboxylate

A mixture of Intermediate 7 (19.2 g, 60 mmol), Intermediate 12 preparedaccording to above procedure and potassium carbonate (16.6 g, 120 mmol)in anhydrous DMF (300 mL) was stirred overnight at RT. The reactionmixture was poured into water (250 mL) and filtered, and the filter cakewas dried under vacuum to afford Intermediate 13 (25 g, 75% yield) asyellow solid. HPLC: t_(R)=2.68 min in 10-80AB_6 min chromatography(Ultimate XB-C18, 3.0*50 mm, 3 um). LCMS: t_(R)=0.792 min in 5-95AB_1.5min chromatography (Welch Xtimate C18, 2.1*30 mm, 3 um), MS (ESI) m/z546.0 [M+H]⁺.

Intermediate 14(±)-4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl-2-methylpiperazine-1-carboxylate

A mixture of Intermediate 13 (25 g, 46 mmol) in a solution ofHCl/dioxane (250 mL, 4M) was stirred for 30 min at RT. The resultingsolid was collected and redissolved in water, and then adjusted to pH=8with saturated NaHCO₃. The precipitate was collected and washed withCH₂Cl₂. The solid was dried under vacuum to give the product (19 g, 93%yield) as yellow solid. HPLC: t_(R)=1.58 min in 10-80AB_6 minchromatography (Ultimate XB-C18, 3.0*50 mm, 3 um). LCMS: t_(R)=0.638 minin 5-95AB_1.5 min chromatography (Welch Xtimate C18, 2.1*30 mm, 3 um),MS (ESI) m/z 445.1 [M+H]⁺.

¹H NMR (CD₃OD 400 MHz): δ 8.44 (s, 1H), 8.08 (s, 1H), 7.60 (t, 1H), 7.39(t, 1H), 7.27-7.20 (m, 2H), 4.41 (s, 1H), 4.00 (s, 3H), 3.08-2.79 (m,4H), 1.43 (brs, 3H).

Example 1 4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate

To a mixture of Intermediate 10 (8 g, 15 mmol, 85% purity) andparaformaldehyde (1 g, 32 mmol) in MeOH (100 mL) was added sodiumcyanoborohydride (2 g, 32 mmol) and the reaction mixture was stirred atRT overnight. The reaction mixture was concentrated in vacuo, theresidue was diluted with water and extracted with EtOAc (3×100 mL). Thecombined organic layers were washed with brine, dried over anhydroussodium sulfate and concentrated under reduced pressure. The crudeproduct was purified by reverse phase preparative HPLC (column: synergi77*250.10 um, gradient: 5-35% B (A=water/0.05% formic acid,B=acetonitrile), flow rate: 140 mL/min). The fraction contained desiredproduct was neutralized with saturated potassium carbonate and extractedwith EtOAc. The combined organic layer was concentrated in vacuo andfreeze-dried to afford Example 1 (4 g, 58% yield for 2 steps) as whitesolid.

LC-MS: t_(R)=1.406 min in 4 min chromatography, MS (ESI) m/z 460.0[M+H]⁺

SFC: t_(R)=1.637 min in 3 min chromatography (Chiralpak AD-3 50*4.6 mmI.D, 3 um), MS (ESI) m/z 460.1 [M+H]⁺

¹H NMR (CDCl₃ 400 MHz): δ 8.76 (s, 1H), 8.53-8.48 (m, 1H), 7.65 (s, 1H),7.44 (brs, 1H), 7.34 (s, 1H), 7.19-7.15 (m, 2H), 4.51-4.50 (m, 1H),4.20-4.05 (m, 1H), 3.99 (s, 3H), 3.50-3.30 (m, 1H), 2.87 (d, 1H), 2.73(d, 1H), 2.35 (s, 3H), 2.35-2.25 (m, 1H), 2.13-2.11 (m, 1H), 1.47 (s,3H).

Example 1, Form A

Form A material was produced by heating Example 1 to 140° C.Approximately 10 mg of Example 1 was placed in an aluminium pan. The panwas heated to 140° C. with the heating rate of 10° C./min usingdifferential scanning calorimetry (DSC) and subsequently cooled to RTunder nitrogen gas.

Form A material was also produced by slow evaporation of Example 1 inIPA. Approximately 10 mg of Example 1 was weighed to a 3-mL vial, 0.25mL of IPA was added to dissolve the solid. After evaporating at RT for24 hours, Example 1 (Form A) was obtained.

Form A material was also produced by slurrying Example 1 in MTBE for 24hours at 50° C. Approximately 10 mg of Example 1 was weighed to a 3-mLvial, 1 mL of MTBE was added and then the suspension was stirred for 24hours at 50° C. to obtain Example 1 (Form A) was obtained.

Form A material was also produced by anti-solvent addition ofEtOAc/heptane. Approximately 10 mg of Example 1 was weighed to a 5-mLvial, 1 mL of EtOAc was added to dissolve the solid and the 4 mL ofanti-solvent heptane was added to the vial slowly. The mixture wasstirred for 24 hours at RT to obtain Example 1 (Form A).

The X-ray powder diffraction spectra for Example 1 (Form A) showed thematerial to be crystalline. The material had a melting point of 192.4°C. (onset).

Example 1, Form E

Approximately 10 mg of Example 1 was weighed to a 5 mL vial, 0.25 mL ofTHF was added to dissolve the solid, then 4 mL of anti-solvent heptanewas added to the vial and the mixture stirred for 24 hours at RT beforethe solid was isolated. The sample (Form E) was determined to becrystalline by XRPD and had a melting point of 194.2° C. (onset).

Example 1, Form I

Approximately 10 mg of Example 1 was weighed to a 3 mL vial, 1 mL of H₂Oto was added the vial and the suspension stirred for 24 hours at 50° C.before the solid was isolated. The sample (Form I) was determined to becrystalline by XRPD and had a melting point of 193.3° C. (onset).

Example 1, Form J

Approximately 10 mg of Example 1 was weighed to a 3 mL vial, 1 mL of H₂Owas added to the vial and the suspension stirred for 24 hours at RTbefore the solid was isolated. The sample (Form J) was determined to becrystalline by XRPD and had a melting point of 193.3° C. (onset).

Example 2 4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate hydrochloride

Example 1 (1.8 g) was dissolved in acetonitrile (5 mL), then 1 N HCl (5mL) was added slowly, the solution was dried by lyophilization to giveExample 2 (1.93 g) as a yellow solid. LC-MS: t_(R)=1.355 min in 4 minchromatography, MS (ESI) m/z 460.1 [M+H]⁺. SFC: t_(R)=1.773 min in 3 minchromatography (Chiralpak AD-3 50*4.6 mm I.D, 3 um), MS (ESI) m/z 460.1[M+H]⁺. ¹H NMR (CD₃OD 400 MHz): δ 8.55 (s, 1H), 8.33-8.16 (m, 1H), 7.56(t, 1H), 7.45 (t, 1H), 7.33 (s, 1H), 7.28-7.20 (m, 1H), 4.81-4.59 (m,1H), 4.52-4.15 (m, 1H), 4.10-3.95 (m, 3H), 3.74-3.48 (m, 3H), 3.35 (br.s., 1H), 3.24-3.09 (m, 1H), 2.97 (s, 3H), 1.54 (br. s., 3H).

Formation of Example 2 Mono-HCl Salt Form A₁

To approximately 10 mg of Example 1 was added 0.35 mL of IPA, followedby 0.217 mL of hydrochloric acid. The solution was sealed tightly with acap and left to stir on a magnetic stirrer plate. During the stirring,some white precipitate was observed. After approximately 24 hours, thesample was separated and dried at RT by vacuum. This form (mono-HCl saltForm A₁) was determined to be crystalline by XRPD and had a meltingpoint of 259.6° C. (onset).

Mono-HCl salt Form A₁ was also produced by reaction crystallization ofExample 1 and hydrochloric acid in EtOH at RT. To approximately 10 mg ofExample 1, was added 0.35 mL of EtOH to dissolve the solid, then 0.217mL of hydrochloric acid was added to the solution. The solution wassealed tightly with a cap and left to stir on a magnetic stirrer plate.During the stirring, some white precipitate was observed. Afterapproximately 24 hours, the sample was separated and dried at RT byvacuum. This form (mono-HCl salt Form A₁) was determined to becrystalline by XRPD and had a melting point of 259.6° C. (onset).

Mono-HCl salt Form A₁ was also produced by reaction crystallization ofExample 1 and hydrochloric acid in acetone at RT. To approximately 10 mgof Example 1, was added 0.35 mL of acetone to dissolve the solid,followed by 0.217 mL of hydrochloric acid. The solution was sealedtightly with a cap and left to stir on a magnetic stirrer plate. Duringthe stirring, some white precipitate was observed. After approximately24 hours, the sample was separated and dried at RT by vacuum. This form(mono-HCl salt Form A₁) was determined to be crystalline by XRPD and hada melting point of 259.6° C. (onset).

Mono-HCl salt Form A₁ was also produced by reaction crystallization ofExample 1 and hydrochloric acid in THF at RT. To approximately 10 mg ofExample 1, was added 0.35 mL of THF to dissolve the solid, then 0.217 mLof hydrochloric acid was added. The solution was sealed tightly with acap and left to stir on a magnetic stirrer plate. During the stirring,some white precipitate was observed. After approximately 24 hours, thesample was separated and dried at RT by vacuum. This form (mono-HCl saltForm A₁) was determined to be crystalline by XRPD and seen to bedifferent to previously seen forms. This material had a melting point of259.6° C. (onset).

Example 3 4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2S)-2,4-dimethylpiperazine-1-carboxylate

A solution of Intermediate 7 (150 mg, 0.47 mmol), Intermediate 11 (1 g,crude) and K₂CO₃ (130 mg, 0.94 mmol) in N,N-dimethyl-formamide (10 mL)was stirred at 30° C. overnight as monitored by LCMS. The solution wasfiltered and purified by reverse phase preparative HPLC (column: ASB150*25 mm*5 um, gradient: 3-28% B (A=water/0.05% HCl, B=acetonitrile),flow rate: 30 mL/min) to give Example 3 (21.0 mg). LC-MS t_(R)=1.156 minin 4 min chromatography, MS (ESI) m/z 460.0 [M+H]⁺ SFC: t_(R)=2.084 minin 3 min chromatography (Chiralpak AD-3 50*4.6 mm I.D, 3 um), MS (ESI)m/z 460.1 [M+H]⁺; ¹H NMR (CD₃OD, 400 MHz): δ8.77 (s, 1H), 8.43 (s, 1H),7.57-7.50 (m, 2H), 7.38 (s, 1H), 7.32-7.28 (m, 1H), 4.51-4.21 (m, 1H),4.10 (s, 3H), 3.77-3.35 (m, 5H), 3.27-3.17 (m, 1H), 2.99 (s, 3H),1.58-1.49 (m, 3H).

Example 4 4-[(3-Chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(±) 2,4-dimethylpiperazine-1-carboxylate

A mixture of Intermediate 14 (1.0 g, 2.0 mmol, 96% purity),paraformaldehyde (200 mg, 6.6 mmol), acetic acid (400 mg, 6.6 mmol) inMeOH (15 mL) was stirred for 2 hours at RT. Sodium cyanoborohydride (400mg, 6.6 mmol) was added. The resulting reaction mixture was stirred foranother 2 hours. The mixture was worked up and purified by reverse phasepreparative HPLC (column: ASB, gradient: 5-30% B (A=water/0.05% HCl,B=acetonitrile), flow rate: 30 mL/min) to afford Example 4 (300 mg, 27%)as white solid. LC-MS t_(R)=1.099 min in 4 min chromatography, MS (ESI)m/z 460.1 [M+H]⁺; ¹H NMR (CD₃OD 400 MHz): δ 8.79 (s, 1H), 8.51 (s, 1H),7.58-7.52 (dd, 2H), 7.45 (s, 1H), 7.34-7.30 (t, 1H), 4.71-4.30 (m, 2H),4.13 (s, 3H), 3.75-3.58 (m, 3H), 3.55-3.42 (m, 1H), 3.27 (s, 1H), 3.02(s, 3H), 1.62-1.53 (m, 3H).

Example 5 Alternative crystalline forms of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate

XRPD Angle 2- XRPD Melting Freebase Theta Intensity point FormPreparation Methods (2θ) (%) (° C.) Form A Amorphous freebase heated to135° C. 23.3, 14.3, 100.00, 192.4 (10° C./min) 9.4 83.70, Slowevaporation: IPA, iPrOAc 78.08 Slurry in heptane, MTBE, DCM/heptane(1/4, v/v), THF/heptane (1/4, v/v), iPrOAc/heptane (1/4, v/v) at RTSlurry in heptane, MTBE, acetone/H₂O (1/4, v/v), MeOH/H₂O (1/4, v/v),EtOH/H₂O (1/4, v/v), THF/H₂O (1/4, v/v), DCM/heptane (1/4, v/v),THF/heptane (1/4, v/v), iPrOAc/heptane (1/4, v/v), EtOH/heptane (1/4,v/v) at 50° C. Anti-solvent addition (solvent/anti- solvent):EtOAc/heptane, DCM/heptane Wet grinding: acetone, EtOAc Form B Slurry inacetone/H₂O (1/4, v/v), MeOH/ 6.3, 3.1, 100.00, N/A H₂O (1/4, v/v),EtOH/H₂O (1/4, v/v), 12.6 52.07, THF/H₂O (1/4, v/v) at RT 35.29Anti-solvent addition (solvent/anti- solvent): MeOH/H₂O, THF/H₂O,Dioxane/H₂O Wet grinding: EtOH/H₂O (1/1, v/v) Form C Slow evaporation:THF 15.6, 8.6, 100.00, N/A Anti-solvent addition (solvent/anti- 13.960.20, solvent): dioxane/hepane 34.59 Form D Slow evaporation: EtOH 7.3,11.4, 100.00, N/A Slurry in EtOH/heptane (1/4, v/v) at RT 21.0 39.48,Anti-solvent addition (solvent/anti- 23.59 solvent): EtOH/MTBE Form EAnti-solvent addition (solvent/anti- 7.3, 13.7, 100.00, 93.0 solvent):THF/heptane 13.4 81.83, 74.07 Form F Slow evaporation: acetone 9.3,16.0, 100.00, N/A 21.6 69.50, 57.55 Form G Slow evaporation: acetone5.1, 7.2, 100.00, N/A Wet grinding: DCM 17.0 12.14, 8.13 Form H Slowevaporation: MeOH 7.7, 21.2, 100.00, N/A Wet grinding: MeOH 19.5 41.70,39.40 Form I Slurry in H₂O at 50° C. 3.5, 7.0, 100.00, 193.3 9.5 41.22,32.57 Form J Slurry in H₂O at RT 7.8, 7.0, 100.00, N/A 4.9 49.36, 45.57

Example 6 Alternative salt forms of4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl(2R)-2,4-dimethylpiperazine-1-carboxylate

XRPD Angle 2- XRPD Melting Salt Theta Intensity point Form PreparationMethods (2θ) (%) (° C.) HCl salt Reaction crystallization of thefreebase 12.3, 13.9, 100.00, 259.6 Form A₁ and hydrochloric acid in IPA,EtOH, 9.3 40.45, acetone or THF at RT 29.34 HCl salt Reactioncrystallization of the freebase 6.6, 13.2, 100.00, N/A Form B₁ andhydrochloric acid in EtOH/H₂O (v/v, 12.6 52.30, 19/1) at RT, thenevaporation 38.68 Sulfate Reaction crystallization of the freebase 19.8,20.4, 100.00, N/A Form A₂ and sulfuric acid in IPA at RT 22.3 36.69,26.58 Sulfate Reaction crystallization of the freebase 7.2, 16.7,100.00, 223.7 Form B₂ and sulfuric acid in EtOH, acetone, THF 14.568.32, or EtOH/H₂O (v/v, 19/1)at RT 45.68 Phosphate Reactioncrystallization of the freebase 7.0, 16.5, 100.0, 206.0 Form A₃ andphosphoric acid in EtOH at RT 22.4 61.81, 29.12 Phosphate Reactioncrystallization of the freebase 5.1, 23.4, 100.00, 177.8 Form B₃ andphosphoric acid in EtOH/H₂O (v/v, 11.9 18.27, 19/1) at RT 16.19 MaleateReaction crystallization of the freebase 4.9, 6.6, 100.00, 108.1 Form A₄and maleic acid in IPA at RT 12.6 93.00, Reaction crystallization of thefreebase 30.51 and maleic acid in acetone at RT, then evaporationMaleate Reaction crystallization of the freebase 6.7, 4.5, 100.00, 120.0Form B₄ and maleic acid in DCM or THF at RT 20.2 26.67, 11.01 MaleateReaction crystallization of the freebase 6.3, 8.5, 100.00, N/A Form C₄and maleic acid in EtOH/H₂O (v/v, 19/1) 10.6 87.86, at RT, thenevaporation 63.25 Tartrate Reaction crystallization of the freebase13.3, 6.6, 100.00, 158.5 Form A₅ and tartaric acid in EtOH or EtOH/H₂O17.6 63.41, (v/v, 19/1) at RT 49.61 Fumarate Reaction crystallization ofthe freebase 6.6, 5.2, 100.00, 212.8 Form A₆ and fumaric acid in acetoneat RT 20.4 51.69, Reaction crystallization of the freebase 29.49 andfumaric acid in IPA at RT, then evaporation Fumarate Reactioncrystallization of the freebase 9.3, 9.8, 100.00, 205.8 Form B₆ andfumaric acid in DCM at RT 26.7 58.74, 54.18 Fumarate Reactioncrystallization of the freebase 7.2, 17.0, 100.00, 199.2 Form C₆ andfumaric acid in EtOH/H₂O (v/v, 6.2 86.58, 19/1) at RT, then evaporation54.86 Citrate Reaction crystallization of the freebase 28.3, 15.2,100.00, 157.9 Form A₇ and citric acid in DCM at RT 22.2 36.42, 26.50

The invention claimed is:
 1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof.
 2. The pharmaceuticallyacceptable salt of the compound as claimed in claim
 1. 3. The compoundof formula (I), or a pharmaceutically acceptable salt thereof, asclaimed in claim 1 in crystalline form.
 4. A pharmaceutical compositionwhich comprises a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, as claimed in claim 1, in association with apharmaceutically-acceptable diluent or carrier.
 5. A compound of formula(I), or a pharmaceutically acceptable salt thereof, as claimed in claim1, in combination with an anti-tumour agent selected from: (i) ananti-CTLA-4 antibody; (ii)6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide or a pharmaceutically acceptable saltthereof; (iii) an anti-PD-L1 antibody; (iv)1-[(1S)-1-(imidazo[1,2-a]pyridin-6-yl)ethyl]-6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazineor a pharmaceutically acceptable salt thereof; (v) an anti-PD-1antibody; or (vi) an OX40 agonist antibody.
 6. A method of treatingnon-small cell lung cancer, in a warm blooded animal in need of suchtreatment which comprises administering to said animal an effectiveamount of a compound of formula (I):

or a pharmaceutically acceptable salt thereof.
 7. The method of claim 6,wherein the non-small cell lung cancer is metastatic non-small cell lungcancer.
 8. The method of claim 7, wherein the metastatic non-small celllung cancer has metastasized to the brain.
 9. The method of claim 7,wherein the metastatic non-small cell lung cancer has metastasized tothe meninges.
 10. The method of claim 6, wherein the compound of formula(I), or the pharmaceutically acceptable salt thereof, is in crystallineform.
 11. The method of claim 6, wherein the compound of formula (I), ora pharmaceutically acceptable salt thereof, is provided in associationwith a pharmaceutically-acceptable diluent or carrier.
 12. The method ofclaim 6, wherein the compound of formula (I), or a pharmaceuticallyacceptable salt thereof, is provided in combination with an anti-tumouragent selected from: (i) an anti-CTLA-4 antibody; (ii)6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide or a pharmaceutically acceptable saltthereof; (iii) an anti-PD-L1 antibody; (iv) 1-[(1S)-1-(imidazo[1,2-a]pyridin-6-yl)ethyl]-6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazineor a pharmaceutically acceptable salt thereof; (v) an anti-PD-1antibody; or (vi) an OX40 agonist antibody.
 13. The method of claim 6,wherein the warm blood animal is man.